The overall aim of this application is to advance PfSEA-1 as a vaccine candidate for falciparum malaria. P. falciparum malaria is a leading cause of morbidity and mortality in developing countries, infecting hundreds of millions of individuals and killing over one million children in sub-Saharan Africa each year. In previous R01 funded studies, we identified the parasite targets of naturally acquired protective human antibodies. We applied our differential, whole proteome screening method using plasma and epidemiologic data from a birth cohort of children living in Tanzania to identify novel Plasmodium falciparum antigens associated with resistance in two-year old children. This work has culminated in a comprehensive, full length Research Article in Science. We discovered Schizont Egress Antigen-1 (PfSEA-1), a 244-kDa parasite antigen which localizes to the schizont/parasitophorous vacuole membrane, Maurer's clefts and the inner leaflet of the RBC membrane in schizont infected RBCs. Antibodies to a central, highly invariant 274 aa region of PfSEA-1 (rPfSEA-1A, aa 810- 1083) decrease parasite replication by 58-75% across three parasite strains compared to controls (all P < 0.009) by arresting schizont rupture. In genetic destabilization experiments, parasites with destabilized PfSEA- 1 had a marked, 4.9 fold defect in parasite replication (P < 0.001). Active vaccination with rPbSEA-1 results in a 3.9 fold reduction in parasitemia and 2 fold longer survival after challenge with P. berghei ANKA parasites (P = 0.001). Children in our Tanzanian cohort experienced a dramatically increased incidence of severe malaria during periods with undetectable anti-PfSEA-1 antibody levels (45 cases/23,806 child weeks) compared to periods with detectable antibody levels (0 cases/1,688 child weeks; adjusted OR 4.4; Type III fixed effects P < 0.01). Adolescents and young adults in our Kenyan cohort with detectible anti-PfSEA-1 antibodies had 50% lower parasitemia compared to individuals without detectible anti-PfSEA-1 antibodies. These results represent the first demonstration that antibodies that specifically block schizont egress can protect against severe malaria in humans and suggest that PfSEA-1 may synergize with vaccines targeting hepatocyte and red cell invasion. Our vaccine discovery program has also identified several known invasion ligands (MSP-4 and MSP-7 3- collectively referred to as MSPs) and PfGARP, a previously unrecognized vaccine candidate found only in falciparum. In in vitro assays, polyclonal anti-MSP-4 inhibited parasite growth by 22% (P < 0.001) and this inhibition increased to 67% when combined with anti-PfSEA-1 mAb (P < 0.001). Antibodies to the highly invariant carboxyl terminal of PfGARP (PfGARP-A, aa 411-673) inhibited parasite growth in vitro by 99% compared to controls (P < 0.001) by arresting trophozoite development. In this application, we will evaluate combinations of these vaccine candidates in multiple adjuvants in murine vaccine trials and the lead combination will be further evaluated in non-human primates. The deliverables from this study will be an adjuvant optimized tri-valent vaccine that targets the entry, intracellular development, and the exit of the parasite from a single, critical parasite stae.